中文摘要

真核生物遭遇逆境（stress）時，細胞會經由訊息傳遞系統來傳遞外界刺激的訊息，以產生適當的生理反應，並進而適應環境的變化。各種不同的外界訊息藉由細胞膜上的受器接收後，會開啟一連串的磷酸化及去磷酸化反應，最後活化轉錄因子，進而促使目標基因表現，以反應外界的訊息。其中MAPK（mitogen-activated protein kinase）訊息傳遞途徑扮演極重要的角色，已經被證實參與許多逆境的訊息傳遞。
在哺乳動物系統中，已有許多證據顯示MAPK參與不同重金屬之訊息傳遞，然而關於植物重金屬訊息傳遞的研究仍相當少，因此，本研究將探討植物細胞在重金屬逆境下之訊息傳遞途徑。為了研究MAPK訊息傳遞途徑是否與植物重金屬逆境相關，我們首先分析重金屬逆境下水稻MAPK基因的表現情形，結果發現分析的三個基因中，OsMAPK2（Accession No. AJ250311）會受重金屬鎘及銅的誘導表現，而OsMAPK3（Accession No. AF241166）及OsMAPK4（Accession No. AJ251330）的表現則沒有明顯增加。因此，我們進一步以西方墨點法及in-gel kinase assay確認水稻MAPK在重金屬逆境下是否具有活性，試驗結果顯示，水稻懸浮細胞及根部在鎘及銅處理1小時後，會誘導40-kD及42-kD MAPK的活化，顯示MAPK途徑可能參與重金屬的訊息傳遞。由於ROS (reactive oxygen species)及鈣離子為重要的二級傳訊者，因此，我們進一步探討ROS及鈣離子與重金屬訊息傳遞途徑之關係。試驗結果顯示，鎘及銅處理會造成水稻根部細胞累積ROS及鈣離子，當以抗氧化劑(GSH及sodium benzoate)及鈣離子螯合劑(EGTA及BAPTA)進行前處理，則可以抑制重金屬所誘導的MAPK活性，顯示重金屬可能是藉由誘導ROS或鈣離子的累積，進而活化MAPK訊息傳遞途徑。為了確認重金屬所誘發之ROS的來源，我們以NADPH oxidase抑制劑DPI (diphenylene iodonium)對水稻根部細胞進行前處理，結果發現DPI只抑制鎘所誘導的MAPK活性，對於銅所誘導的MAPK活性則無顯著影響；此外，以鎘及銅處理NADPH oxidase突變株，也發現只有鎘處理突變株時，MAPK活性才會受到抑制，上述結果顯示，鎘會透過活化NADPH oxidase以產生ROS，而銅則可能藉由其他的機制。除了鈣離子以外，我們也證實CDPK (calcium-dependent protein kinase)可能位於鎘及銅所活化的MAPK途徑之上游。由於G protein及PI3 kinase參與許多逆境訊息傳遞，本研究也利用G protein突變株及PI3 kinase抑制劑，證實G protein及PI3 kinase可能參與鎘及銅的訊息傳遞，並作用於MAPK途徑的上游。
為了深入了解MAPK途徑於重金屬逆境下所扮演之角色，我們以23個不同的水稻品種進行對鎘逆境反應之測試，並利用已知的耐鎘品種台農六十七號(TNG67)及不耐鎘品種台中再來一號(TN1)作為對照，結果篩選出台農七十一號(TNG71)、台梗四號(TK4)、台梗十一號(TK11)、台南十一號(TN11)、桃園一號(TY1)及台南糯十號(TNW10)可能為耐鎘品種，而桃園糯二號(TYW2)、台秈二號(TS2)、台中秈十號(TCS10)及台中秈十四號(TCS14)可能為不耐鎘品種。進一步以p-ERK抗體進行西方墨點法分析，發現於耐鎘品種中，42-kD MAPK磷酸化程度均明顯較不耐鎘品種高，顯示42-kD MAPK可能扮演提供水稻重金屬耐性的角色。

ABSTRACT

Protein phosphorylation and dephosphorylation are important in the regulation of physiological status and gene expression in response to extracellular stimuli. Mitogen-activated protein kinase (MAPK) cascade is one of the major pathways transfer information from sensors to cellular responses in all eukaryotes. The MAPK cascade is a protein module, which is composed of MAPK, MAPKK (MAPK kinases) and MAPKKK (MAPKK kinase). A large number of genes encoding MAPK pathway components have been identified in model plants and demonstrated their roles in developmental and stress signal transduction.
There are ample evidences that exposure to excess concentrations of heavy metals activate MAPK pathways in animals. However, little is known about how plants response to heavy metal stress. To characterize signalling pathways involved in heavy metal-induced stress responses, we initially examine whether plant MAPKs are also involved in this process. Three rice MAPK genes, OsMAPK2 (Accession No. AJ250311), OsMAPK3 (Accession No. AF241166) and OsMAPK4 (Accession No. AJ251330), have been isolated in the previous studies of our laboratory and been demonstrated that their gene expressions are associated with environment stresses. In this study, we found that Cd and Cu induced OsMAPK2 gene expression and 40- and 42-kD MAP kinase activation in rice. However, the regulation of MAPK pathways may mainly depend on posttranslational phosphorylation during the early process of heavy metal signal transduction in rice.
To find out the heavy metal-induced signalling pathways, we further investigated the roles of reactive oxygen species (ROS) and calcium in Cd- and Cu-induced MAP kinase activation. The results indicated that Cd and Cu rapidly induced ROS and calcium accumulations in the cytosol, and these increased ROS and calcium subsequently activated MAP kinases. However, redox-dependent activation of MAP kinases by Cd and Cu may occur through distinct ROS-generating systems. Furthermore, calcium-dependent protein kinase (CDPK), an important downstream component of calcium, plays a role in MAP kinase activation by Cd and Cu. Moreover, heterotrimeric G protein and PI3 kinase are also involved in heavy metal signal transduction.
Finally, in order to better understand the roles of MAP kinases in heavy metal stress responses, we identified Cd-tolerant and Cd-sensitive cultivars and analyzed the Cd- and Cu-induced MAP kinase activities in these cultivars. The 42-kD MAP kinase activities are apparent in Cd-tolerant cultivars than in Cd-sensitive cultivars. Therefore, the Cd-induced 42-kD MAP kinase activation may confer Cd tolerance in rice plants.

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